Establishment of a LAMP Assay for Rapid Detecting Diaporthe citri on Pomelo
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摘要:
目的 建立一种蜜柚间座壳黑点病菌快速、简便、灵敏的LAMP（Loopmediated isothermal amplification）可视化检测方法，在植株表现症状之前或初期实现病原菌的监测，为病害的早期监测、诊断及防治提供依据。 方法 以EF-1a（Elongation factor-1α）基因序列为靶序列，设计了一套LAMP特异性引物；以蜜柚黑点病菌DNA为模板，优化反应温度和反应时间，建立LAMP检测反应体系，开展特异性和灵敏度验证及田间病株检测。 结果 建立的LAMP反应体系可特异有效地检测出蜜柚黑点病菌。该体系最佳反应温度、反应时间分别为65 ℃和60 min。灵敏度验证结果表明，LAMP检测最小质量浓度为10 fg·μL−1。采用LAMP法对15份疑似蜜柚黑点病田间样本进行快速检测，检出率为100%。 结论 本研究建立的蜜柚间座壳黑点病菌LAMP快速检测方法不仅特异性强、灵敏度高，且检测结果可视，可用于田间黑点病菌的实时监测和快速检测。 -
关键词:
- 蜜柚 /
- 黑点病 /
- 间座壳菌 /
- 环介导等温核酸扩增技术 /
- 检测
Abstract:Objective A loop-mediated isothermal amplification (LAMP) method for visual, rapid, and accurate detection of Diaporthe citri on pomelos in early stage of black spot disease was developed. Method Based on the elongation factor-1α (EF-1α) sequence of D. citri, a set of LAMP primers was designed. Template DNA from the infected leaves was used to establish the optimal temperature and time for the LAMP operation. Assay specificity and sensitivity were verified by positive detection of the infected plants in the field. Result The newly established LAMP method could effectively and specifically detect D. citri on pomelo at the optimal temperature of 65 ℃ with 60 m for the reaction time. The detection limit of the assay on D. citri was 10 fg·μL−1. On 15 field samples with the typical black spot symptoms, a 100% positive detection rate was achieved by the LAMP assay. Conclusion The established visual and rapid LAMP assay demonstrated a high specificity, sensitivity, and perfect detection of D. citri on diseased pomelo. It was considered appropriate for field application. -
Key words:
- Pomelo /
- black spot /
- Diaporthe citri /
- loop-mediated isothermal amplification /
- detection
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图 1 LAMP反应最佳温度筛选
LAMP 产物颜色反应。1：60 ℃；2：61 ℃；3：62 ℃；4：63 ℃；5：64 ℃；6：65 ℃；7：66 ℃；8：67 ℃。
Figure 1. Optimal temperature for LAMP assay
Color reaction of LAMP products. 1: 60 ℃; 2: 61 ℃; 3: 62 ℃; 4: 63 ℃; 5: 64 ℃; 6: 65 ℃; 7: 66 ℃; 8: 67 ℃.
图 2 LAMP反应最佳时间测定
LAMP 产物颜色反应。1：30 min；2：40 min；3：50 min；4：60 min；5：70 min。
Figure 2. Optimal time for LAMP assay
Color reaction of LAMP products. 1: 30 min; 2: 40 min; 3: 40 min; 4: 60 min; 5: 70 min.
图 3 LAMP检测特异性验证（a：扩增曲线；b：LAMP 产物）
A1：间座壳菌；A2：蜜柚炭疽病菌；A3：细极链格孢菌； A4：禾谷镰孢菌；A5：大斑凸脐蠕孢 ；A6：直立帚枝杆孢；A7：蜜柚黑斑病菌 ；A8：空白对照。
Figure 3. Specificity of LAMP assay (a: primary curve; b: LAMP products)
A1: D. citri; A2: Colletotrichum gloeosporioides; A3: Alternaria tenuissima; A4: Fusarium graminearum; A5: Exserohilum turcicum; A6: Sarocladium strictum; A7: Phyllosticta citriasiana; A8: blank CK.
图 4 LAMP灵敏度检测验证（a：扩增曲线；b：LAMP 产物）
A1-8：DNA 质量浓度分别为 1 ng·μL−1、100 pg·μL−1、10 pg·μL−1、1 pg·μL−1、100 fg·μL−1、10 fg·μL−1、1 fg·μL−1、0.1 fg·μL−1。
Figure 4. Sensitivity of LAMP assay (a: primary curve; b: LAMP products)
A1-8: amplified products using DNA at concentrations of 1 ng·μL−1, 100 pg·μL−1, 10 pg·μL−1, 1 pg·μL−1, 100 fg·μL−1, 10 fg·μL−1, 1 fg·μL−1, 0.1 fg·μL−1.
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